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目的原核表达并纯化翻译调控肿瘤蛋白(Translationally controlled tumor protein,TCTP)。方法以人乳腺癌细胞系MCF-7总RNA为模板,PCR扩增TCTP基因,克隆至原核表达载体pET-28a(+)中,转化大肠杆菌BL21(DE3),IPTG诱导表达。His Trap FF层析柱纯化重组蛋白后,进行Western blot鉴定。结果克隆的目的基因序列正确,未发生碱基突变;重组表达质粒经双酶切鉴定构建正确;表达的重组蛋白相对分子质量约为20 000,主要以包涵体形式表达;纯化后纯度为80%,可与兔抗人TCTP多克隆抗体特异性结合。结论已成功在大肠杆菌BL21(DE3)中表达并纯化了TCTP,为进一步研究其在肿瘤等疾病发生、发展及治疗中的作用奠定了基础。
Objective To express and purify translationally controlled tumor protein (TCTP) in prokaryotic cells. Methods The TCTP gene was amplified by PCR from human breast cancer cell line MCF-7 and cloned into prokaryotic expression vector pET-28a (+). The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. His Trap FF column purification of recombinant protein, Western blot identification. Results The sequence of the cloned gene was correct and no base mutation was observed. The recombinant plasmid was identified by double enzyme digestion. The expressed recombinant protein was about 20,000 in molecular weight and mainly expressed in inclusion bodies. The purity was 80% , Can be combined with rabbit anti-human TCTP polyclonal antibody. Conclusion The expression and purification of TCTP in Escherichia coli BL21 (DE3) has been successfully established, which lays the foundation for the further study of its role in the occurrence, development and treatment of diseases such as cancer.